This invention pertains to the fields of immunodiagnostics and immunotherapeutics. In particular, this invention pertains to the discovery of novel human antibodies that specifically bind to c-erbB-2, and to chimeric molecules containing these antibodies.
Conventional cancer chemotherapeutic agents cannot distinguish between normal cells and tumor cells and hence damage and kill normal proliferating tissues. One approach to reduce this toxic side effect is to specifically target the chemotherapeutic agent to the tumor. This is the rationale behind the development of immunotoxins, chimeric molecules composed of an antibody either chemically conjugated or fused to a toxin that binds specifically to antigens on the surface of a tumor cell thereby killing or inhibiting the growth of the cell (Frankel et al. Ann. Rev. Med., 37: 127 (1986)). The majority of immunotoxins prepared to date, have been made using murine monoclonal antibodies Nabs) that exhibit specificity for tumor cells. Immunotoxins made from Mabs demonstrate relatively selective killing of tumor cells in vitro and tumor regression in animal models (id.).
Despite these promising results, the use of immunotoxins in humans has been limited by toxicity, immunogenicity and a failure to identify highly specific tumor antigens (Byers et al. Cancer Res., 49: 6153). Nonspecific toxicity results from the failure of the monoclonal antibody to bind specifically and with high affinity to tumor cells. As a result, nonspecific cell killing occurs. In addition, the foreign immunotoxin molecule elicits a strong immune response in humans. The immunogenicity of the toxin portion of the immunotoxin has recently been overcome by using the human analog of RNase (Rybak et al. Proc. Nat. Acad. Sci., USA, 89: 3165 (1992)). The murine antibody portion, however, is still significantly immunogenic (Sawler et al., J. Immunol., 135: 1530 (1985)).
Immunogenicity could be avoided and toxicity reduced if high affinity tumor specific human antibodies were available. However, the production of human monoclonal antibodies using conventional hybridoma technology has proven extremely difficult (James et al., J. Immunol. Meth., 100: 5 (1987)). Furthermore, the paucity of purified tumor-specific antigens makes it necessary to immunize with intact tumor cells or partially purified antigen. Most of the antibodies produced react with antigens which are also common to normal cells and are therefore unsuitable for use as tumor-specific targeting molecules.
This invention provides novel human antibodies that specifically bind to the extracellular domain of the c-erbB-2 protein product of the HER2/neu oncogene. This antigen (marker) is overexpressed on many cancers (e.g. carcinomas) and thus the antibodies of the present invention specifically bind to tumor cells that express c-erbB-2.
In a preferred embodiment, the antibody is a C6 antibody derived from the sFv antibody C6.5. The antibody may contain a variable heavy chain, a variable light chain, or both a variable heavy and variable light chain of C6.5 or its derivatives. In addition the antibody may contain a variable heavy chain, a variable light chain or both a variable heavy and variable light chain of C6.5 in which one or more of the variable heavy or variable light complementarity determining regions (CDR1, CDR2 or CDR3) has been altered (e.g., mutated). Particularly preferred CDR variants are listed in the specification and in Examples 1, 2 and 3. Particularly preferred C6 antibodies include C6.5, C6ML3-14, C6L-1 and C6MH3-B1. In various preferred embodiments, these antibodies are single chain antibodies (sFv also known as scFv) comprising a variable heavy chain joined to a variable light chain either directly or through a peptide linker. Other preferred embodiments of the C6 antibodies and C6.5, C6ML3-14, C6L1, and C6MH3-B1, in particular, include Fab, the dimer (Fabxe2x80x2)2, and the dimer (sFvxe2x80x2)2. Particularly preferred (sFvxe2x80x2)2 dimers are fusion proteins where the Sfvxe2x80x2 components are joined through a peptide linkage or through a peptide (G,S). Still other preferred C6 antibodies include an antibody selected from the group consisting of an antibody having a VL domain with one of the amino acid sequences shown in Table 10, an antibody having a VH domain with one of the amino acid sequences shown in Table 12, an antibody having a VL CDR3 domain having one of the amino acid sequences shown in Tables 4, 15, and 16, and an antibody having a VH CDR3 domain having one of the amino acid sequences shown in Tables 13 and 14. Other preferred embodiments are to be found replete throughout the specification.
In a particularly preferred embodiment, the C6 antibody has a Kd ranging from about 1.6xc3x9710xe2x88x928 to about 1xc3x9710xe2x88x9212 M in SK-BR-3 cells using Scatchard analysis or as measured against purified c-erbB-2 by surface plasmon resonance in a BIAcore.
In another embodiment the present invention provides for nucleic acids that encode any of the above-described C6 antibodies. The invention also provides for nucleic acids that encode the amino acid sequences of C6.5, C6ML3-14, C6L1, C6MH3-B1, or any of the other amino acid sequences encoding C6 antibodies and described in Example 1, 2 or 3. In addition this invention provides for nucleic acid sequences encoding any of these amino acid sequences having conservative amino acid substitutions.
In still another embodiment, this invention provides for proteins comprising one or more complementarity determining regions selected from the group consisting of the complementarity determining regions of Tables 10, 12, 13, 14, 15, and 16 and of any of the examples, in particular of Examples 1, 2 or 3. Other particularly preferred antibodies include any of the antibodies expressed by the clones described herein.
In still yet another embodiment, this invention provides for cells comprising a recombinant nucleic acid which is any of the above described nucleic acids.
This invention also provides for chimeric molecules that specifically bind a tumor cell bearing c-erbB-2. The chimeric molecule comprises an effector molecule joined to any of the above-described C6 antibodies. In a preferred embodiment, the effector molecule is selected from the group consisting of a cytotoxin (e.g. PE, DT, Ricin A, etc.), a label, a radionuclide, a drug, a liposome, a ligand, an antibody, and an antigen binding domaine). The C6 antibody may be chemically conjugated to the effector molecule or the chimeric molecule may be expressed as a fusion protein.
This invention provides for methods of making C6 antibodies. One method proceeds by i) providing a phage library presenting a C6.5 variable heavy chain and a multiplicity of human variable light chains; ii) panning the phage library on c-erbB-2; and iii) isolating phage that specifically bind c-erb2. This method optionally further includes iv) providing a phage library presenting the variable light chain of the phage isolated in step iii and a multiplicity of human variable heavy chains; v) panning the phage library on c-erbB-2; and vi) isolating phage that specifically bind c-erbB-2.
Another method for making a C6 antibody proceeds by i) providing a phage library presenting a C6.5 variable light chain and a multiplicity of human variable heavy chains; ii) panning the phage library on c-erbB-2; and iii) isolating phage that specifically bind c-erbB-2.
Yet another method for making a C6 antibody involves i) providing a phage library presenting a C6.5 variable light and a C6.5 variable heavy chain encoded by a nucleic acid variable in the sequence encoding CDR1, CDR2 or CDR3 such that each phage displays a different CDR; ii) panning the phage library on c-erbB-2; and isolating the phage that specifically bind c-erbB-2.
This invention also provides a method for impairing growth of tumor cells bearing c-erbB-2. This method involves contacting the tumor with a chimeric molecule comprising a cytotoxin attached to a human C6 antibody that specifically binds c-erbB-2.
Finally, this invention provides a method for detecting tumor cells bearing c-erbB-2. This method involves contacting the biological samples derived from a tumor with a chimeric molecule comprising a label attached to a human C6 antibody that specifically binds c-erbB-2.
Definitions
The following abbreviations are used herein: AMP, ampicillin; c-erbB-2 ECD, extracellular domain of c-erbB-2; CDR, complementarity determining region; ELISA, enzyme lined immunosorbent assay; FACS, fluorescence activated cell sorter; FR, framework region; Glu, glucose; HBS, hepes buffered saline, 10 mM hepes, 150 mM NaCl, pH 7.4; IMAC, immobilized metal affinity chromatography; kon, association rate constant; koff, dissociation rate constant; MPBS, skimmed milk powder in PBS; MTPBS, skimmed milk powder in TPBS; PBS, phosphate buffered saline, 25 mM NaH2PO4, 125 mM NaCl, pH 7.0; PCR, polymerase chain reaction; RU, resonance units; scFv or sFv, single-chain Fv fragment; sFvxe2x80x2: Fv containing cysteine; TPBS, 0.05% v/v Tween 20 in PBS; SPR, surface plasmon resonance; Vk, immunoglobulin kappa light chain variable region; VI, immunoglobulin lambda light chain variable region; VL, immunoglobulin light chain variable region; VH, immunoglobulin heavy chain variable region; wt, wild type.
As used herein, an xe2x80x9cantibodyxe2x80x9d refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25 kD) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VI) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)xe2x80x22, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)xe2x80x22 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fabxe2x80x2)2 dimer into an Fabxe2x80x2 monomer. The Fabxe2x80x2 monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fabxe2x80x2 fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Preferred antibodies include single chain antibodies, more preferably single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide liner) to form a continuous polypeptide.
An xe2x80x9cantigen-binding sitexe2x80x9d or xe2x80x9cbinding portionxe2x80x9d refers to the part of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (xe2x80x9cVxe2x80x9d) regions of the heavy (xe2x80x9cHxe2x80x9d) and light (xe2x80x9cLxe2x80x9d) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as xe2x80x9chypervariable regionsxe2x80x9d which are interposed between more conserved flanking stretches known as xe2x80x9cframework regionsxe2x80x9d or xe2x80x9cFRsxe2x80x9d. Thus, the term xe2x80x9cFRxe2x80x9d refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen binding xe2x80x9csurfacexe2x80x9d. This surface mediates recognition and binding of the target antigen. The three hypervariable regions of each of the heavy and light chains are referred to as xe2x80x9ccomplementarity determining regionsxe2x80x9d or xe2x80x9cCDRsxe2x80x9d and are characterized, for example by Kabat et al. Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, Md. (1987).
As used herein, the terms xe2x80x9cimmunological bindingxe2x80x9d and xe2x80x9cimmunological binding propertiesxe2x80x9d refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the xe2x80x9con rate constantxe2x80x9d (kon) and the xe2x80x9coff rate constantxe2x80x9d (koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of koff/kon enables cancellation of all parameters not related to affinity and is thus equal to the dissociation constant Kd. See, generals, Davies et al. Ann. Rev. Biochem., 59: 439-473 (1990).
The term xe2x80x9cC6 antibodyxe2x80x9d, as used herein refers to antibodies derived from C6.5 whose sequence is expressly provided herein. C6 antibodies preferably have a binding affinity of about 1.6xc3x9710xe2x88x928 or better and are preferably derived by screening (for affinity to c-erbB-2) a phage display library in which a known C6 variable heavy (VH) chain is expressed in combination with a multiplicity of variable light (VI) chains or conversely a known C6 variable light chain is expressed in combination with a multiplicity of variable heavy (VH) chains. C6 antibodies also include those antibodies produced by the introduction of mutations into the variable heavy or variable light complementarity determining regions (CDR1, CDR2 or CDR3) as described herein. Finally C6 antibodies include those antibodies produced by any combination of these modification methods as applied to C6.5 and its derivatives.
A single chain Fv (xe2x80x9csFvxe2x80x9d or xe2x80x9cscFvxe2x80x9d) polypeptide is a covalently linked VH::VL heterodimer which may be expressed from a nucleic acid including VH- and VL-encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al. Proc. Nat. Acad. Sci. USA, 85: 5879-5883 (1988). A number of structures for converting the naturally aggregatedxe2x80x94but chemically separated light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g. U.S. Pat. Nos. 5,091,513 and 5,132,405 and 4,956,778.
In one class of embodiments, recombinant design methods can be used to develop suitable chemical structures (inkers) for converting two naturally associatedxe2x80x94but chemically separatexe2x80x94heavy and light polypeptide chains from an antibody variable region into a sFv molecule which will fold into a three-dimensional structure that is substantially similar to native antibody structure.
Design criteria include determination of the appropriate length to span the distance between the C-terminal of one chain and the N-terminal of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Such methods have been described in the art. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.
In this regard, the first general step of linker design involves identification of plausible sites to be linked. Appropriate linkage sites on each of the VH and VL polypeptide domains include those which will result in the minimum loss of residues from the polypeptide domains, and which will necessitate a linker comprising a minimum number of residues consistent with the need for molecule stability. A pair of sites defines a xe2x80x9cgapxe2x80x9d to be linked. Linkers connecting the C-terminus of one domain to the N-terminus of the next generally comprise hydrophilic amino acids which assume an unstructured configuration in physiological solutions and preferably are free of residues having large side groups which might interfere with proper folding of the VH and VL chains. Thus, suitable linkers under the invention generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility. One particular linker under the invention has the amino acid sequence [(Gly)4Ser]3 (SEQ ID NO:1). Another particularly preferred linker has the amino acid sequence comprising 2 or 3 repeats of [(Ser)4Gly] (SEQ ID NO:2) such as [(Ser)4Gly]3 (SEQ ID NO:3). Nucleotide sequences encoding such linker moieties can be readily provided using various oligonucleotide synthesis techniques known in the art. See, e.g., Sambrook, supra.
The phrase xe2x80x9cspecifically binds to a proteinxe2x80x9d or xe2x80x9cspecifically immunoreactive withxe2x80x9d, when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, C6 antibodies can be raised to the c-erbB-2 protein that bind c-erbB-2 and not to other proteins present in a tissue sample. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
A chimeric molecule is a molecule in which two or more molecules that exist separately in their native state are joined together to form a single molecule having the desired functionality of all of its constituent molecules. While the chimeric molecule may be prepared by covalently linking two molecules each synthesized separately, one of skill in the art will appreciate that where the chimeric molecule is a fusion protein, the chimera may be prepared de novo as a single xe2x80x9cjoinedxe2x80x9d molecule.
The term xe2x80x9cconservative substitutionxe2x80x9d is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine A), Tyrosine (Y), Tryptophan (W).